Camptothecin (CPT) has been shown to inhibit the growth of a variety of animal and human tumors. Camptothecin and its related congeners display a unique mechanism of action: they stabilize the covalent binding of the enzyme topoisomerase I (topo I), an intranuclear enzyme that is overexpressed in a variety of tumor lines, to DNA. This drug/enzyme/DNA complex leads to reversible, single strand nicks that, according to the fork collision model, are converted to irreversible and lethal double strand DNA breaks during replication. Therefore, due to the mechanism of its cytotoxicity, CPT is S-phase specific, indicating that it is only toxic to cells that are undergoing DNA synthesis. Rapidly replicating cells like cancerous cells spend more time in the S-phase relative to healthy tissues. Thus, the overexpression of topo I combined with the faster rate of cell replication provide a limited basis for selectivity via which camptothecins can effect cytoxicity on cancerous cells rather than healthy host tissues. It is important to note that due to the S-phase specificity of the camptothecins, optimal inhibition of topo I requires continuous exposure to the camptothecin agent.
A closed alpha-hydroxy lactone (E) ring of CPT is an essential structural feature. An intact ring is necessary for the diffusion of the electroneutral form of the drug across membrane barriers and into cells by passive transport and, directly relevant to its in vivo anti-tumor potency, is required for the successful interaction of CPT with the topoisomerase I target. This essential lactone pharmacophore hydrolyzes under physiological conditions (pH 7 or above) and, therefore, the drug can exist in two distinct forms: 1) the biologically active, ring-closed lactone form; and 2) the biologically-inactive, ring-open carboxylate form of the parent drug. Unfortunately, under physiological conditions the drug equilibrium favors hydrolysis and, accordingly, the carboxylate form of the camptothecin drug persists. The labile nature of this alpha-hydroxy lactone pharmacophore has significantly compromised the clinical utility of the camptothecins, as continuous exposures to the active lactone form are requisite for efficacy purposes.
In human blood and tissues, the camptothecins exist in a equilibrium of active lactone form vs. inactive carboxylate form and the directionality of this equilibrium can be greatly affected by the presence of human serum albumin (HSA). Time-resolved fluorescence spectroscopic measurements taken on the intensely fluorescent camptothecin lactone and camptothecin carboxylate species have provided direct information on the differential nature of these interactions with HSA. The lactone form of camptothecin binds to HSA with moderate affinity yet the carboxylate form of camptothecin binds tightly to HSA, displaying a 150-fold enhancement in its affinity for this highly abundant serum protein. Thus, when the lactone form of camptothecin is added to a solution containing HSA, the preferential binding of the carboxylate form to HSA drives the chemical equilibrium to the right, resulting in the lactone ring hydrolyzing more rapidly and completely than when camptothecin is in an aqueous solution without HSA. In turn, this effect has negatively impacted the topoisomerase I inhibitory activity of many camptothecins and, by extension, negatively affects their clinical utility.
The important role that HSA plays in the stability of the camptothecins varies relative to drug structure. For drugs such as camptothecin and 9-aminocamptothecin, HSA functions as a biological sink for the carboxylate form. As a result, in whole human blood, 5.3% of camptothecin and only 0.5% of 9-aminocamptothecin remain in the lactone form at equilibrium. In contrast, A, B-ring substitutions of CPT, specifically at the 7- and 10-positions, can inhibit the preferential binding interactions between the camptothecin carboxylate and HSA. Accordingly, camptothecin congeners such as topotecan and SN-38, the biologically active form of the prodrug CPT-11, display lactone levels at equilibrium of 11.9% and 19.5%, respectively. Ultimately, by modulating the circulatory and tissue levels of free and active camptothecin drug, HSA can negatively impact the anti-cancer efficacy of the camptothecin agent.
The effect of serum albumins on camptothecins also differs markedly between lower vertebrates and humans and this variance has obscured the judicious selection of analogs for advancement to clinical trials. These interspecies difference have lead to significant anomalies when the data from animal models and clinical studies are compared. In particular, 9-aminocamptothecin has displayed striking activity in murine models bearing brain tumors. However, the pharmacokinetics of 9-aminocamptothecin in mice are quite different from those in humans; notably, 9-aminocamptothecin lactone levels are approximately 100-fold higher in murine blood relative to human blood. This discrepancy is due to the reduced binding of the carboxylate form of 9-aminocamptothecin to murine albumin. The logical extension of this finding is that approximately 100-fold more free lactone, which is able to cross cell membranes or the blood-brain barrier, is present in the mouse than it is in humans. The clinical relevance of this interspecies variation is underscored by a recent trial: 99 brain cancer patients were treated intravenously with 9-aminocamptothecin; the therapy was grossly ineffective (one partial responder) due to the likelihood that 99.5% of the drug was in the carboxylate form, bound to HSA and unable to transverse the blood-brain barrier.
The inherent blood instability of camptothecin has resulted in an extensive research effort to surmount the problem. Efforts to realize a blood stable camptothecin agent with potent anti-tumor activity have been primarily focused on formulation, such as liposomal preparations of the drug, and rational drug design, such as the development of the class of beta-hydroxy lactone camptothecins known as the homocamptothecins. The work described herein describes a third approach to maintaining a potent and more blood stable camptothecin congener: the modulation of camptothecin drug binding to HSA by implementing competing molecules that also bind HSA.
The camptothecins are not unique in their ability to bind albumin, as a variety of small molecules interact with this protein. A relatively large protein, 67 kD, albumin is distributed both in the plasma and in the interstitial fluid. Being one of the most abundant plasma proteins, its circulatory level ranges from 35 to 50 mg/ml (approximately 0.6 mM). The principal biological function of HSA is to maintain colloid osmotic pressure in the vascular system and to transport fatty acids and bilirubin. However, by hydrophobic and/or ionic interactions, a variety of small molecules bind tightly to albumin. Electroneutral and basic drugs may bind to albumin by hydrophobic binding interactions, and, as albumin has a net cationic charge, anionic drugs bind avidly to albumin via electrostatic interactions. Albumin possesses two well-characterized binding pockets, as well as other general binding sites. Site I is known as the warfarin binding site, which also binds drugs such as phenylbutazone, sulfonamides, phenytoin, and valproic acid. Site 11 is referred to as the diazepam site, which is also the binding site for benzodiazepines, tryptophan, ibuprofen, naproxen, octanoic acid, clofibric, iopanice, probenecid, semi-synthetic penicillins and medium chain fatty acids. Other general binding sites include sites for bilirubin, digitoxin and a variety of fatty acids. Recent x-ray crystallography and competition data obtained by the present inventors reveal that camptothecin carboxylate preferentially associates with a characterized drug binding site in subdomain IB, which overlaps with one of the main long-chain fatty acid binding sites, protoporphyrin and other drugs and compounds, although it possesses secondary affinity to binding sites I and II. Interestingly, in vivo small molecule binding to albumin is saturable at therapeutically relevant drug levels.
The ability of human serum albumin to avidly bind to a variety of small molecules offers the possibility of competitively attenuating the negative effects human serum albumin on the in vivo anti-cancer and/or anti-HIV activity of camptothecin compounds and numerous other compounds such as camptothecin that have extremely high binding affinity for human serum albumin.
However, no prior methods have recognized or attempted to deal with the problem caused by the human serum albumin binding activity, and thus methods and compositions are needed which can attenuate the negative effects of human serum albumin on the stability of compounds such as camptothecin compounds, e.g., camptothecin or 9-aminocamptothecin, and other compounds or drugs, such as protease inhibitors, which have a high affinity for human serum albumin.